RNA Microinjection Buffer and Cas9 mRNA Reagents

Materials

  • in vitro transcribed mRNA or RNA
  • Oligonucleotide resuspended in RNAse free water
  • DNA donor plasmid prepared with an endotoxin free plasmid kit
  • 0.02 µm Anotop 10 Syringe Filters (Whatman Cat. no. 6809-1002) — Do NOT filter nucleic acid solutions through the Anotop filters, the 0.02 µm pores will trap nucleic acids and remove them from solution
  • Millipore dialysis filter (Millipore #VMWP02500, pore size 0.05 µm)
  • Sterile 10 ml syringe
  • Sterile 10cm Petri dish
  • Pipetman
  • RNAse Free Pipet Tips
  • Sterile 1.5 ml Microtubes
  • Sterile 15 ml tube
  • Sterile 50 ml tube
  • Sterile 10 ml pipets
  • 1 M Tris-HCL, pH 7.4 (Sigma Cat. no. T263)
  • 0.5 M EDTA (Sigma Cat. no. E7889)
  • Nuclease Free Water (Sigma cat. no. W4502)

 

Procedure

RNAse Free Microinjection Buffer (10 mM Tris-HCl, pH 7.4, 0.25 mM EDTA)

For 10 ml, mix together:

  • 9.9 ml water
  • 0.1 ml Tris-HCl, pH 7.4
  • 0.005 ml EDTA

For 20 ml, mix together:

  • 19.8 ml water
  • 0.2 ml Tris-HCl, pH 7.4
  • 0.010 ml EDTA

Wash 0.02 µm filter with 1 ml buffer – discard wash
Filter remaining buffer through filter for use as dialysis buffer or for use as microinjection buffer.

 

mRNA/sgRNA/oligo/plasmid Donor Preparation for Microinjection

The Transgenic Core has used Cas9, TALENs, and zinc finger nucleases to target more then 30 genes in mice and rats. The majority of projects have been gene disruptions caused by non-homologous endjoining. Eight projects used donors to introduce genetic changes by homologous recombination. The suggestions below are based our successful experience.

1. mRNA and sgRNA are transcribed in vitro with kits and procedures as described (Geurts et al, 2009, Wefers et al., 2013, Yang et al., 2013). Nuclease reagents typically clog glass needles used to microinject reagents into fertilized eggs. Clogged needles lead to decreased egg survival and lower yields of genetically modified mice or rats. To reduce clogging and improve outcomes, the Transgenic Core asks that wash buffers and elution buffers used in mRNA and sgRNA purification be pre-filtered through 0.02 um filters. Do not pass nucleic acid solutions through the filters. The pore size on the filters is small enough to trap nucleic acids. Alternatively Cas9 mRNA or Cas9 protein solutions may be purchased from commercial vendors. Whether Cas9 reagents are prepared by the submitting laboratory or obtained from a third party, the Transgenic Core recommends the use of quality control assays to demonstrate activity (i.e. in vitro translation of mRNA into Cas9 protein, Western blotting of translation product, and Cas9 protein detection with antibodies).

2. Plasmid DNA donors should be purified with endotoxin free kits. Pre-filter wash buffers and elution buffers with through 0.02 um filters. Do not pass nucleic acid solutions through the filters. The pore size on the filters is small enough to trap nucleic acids. Kits that are based on columns often produce particulate contaminated DNA as shown by Montigny et al. (2003). We have observed reduced clogging when DNA is purified plasmid or BAC DNA with methods that don’t rely on columns for plasmid DNA purification (such as the Epicentre BACMAX kit).

3. Oligonucleotide donors are subjected to spot dialysis to remove embryotoxic chemicals. Follow standard guidelines for working with RNA to protect against later RNA degradation.

3.1. Fill a 10cm or 15cm Petri dish with nuclease free microinjection buffer. Place a Millipore dialysis filter on the surface of the buffer so that it floats (place the filter shiny side up).

3. 2. Carefully spot the nucleic acid solution into the center of the filter. Replace the Petri dish lid. Dialyze for 30-60 minutes. Up to 200 ul can be placed on a filter without losing it to the buffer. Leave the dialysis to proceed quietly without any shaking or movement. Do not let the dialysis to go more than 3 hours; otherwise the drop might begin to evaporate.

3. 3. Carefully Pipette off the solution. Place the tip in the middle of the droplet and carefully aspirate as much as possible without stopping. Transfer the nucleic acid solution to a sterile microtube. Quantitate and store at – 80°C. Recoveries between 50-70% of the original volume are normal. The rest remains attached as a very thin liquid layer onto the surface of the filter and is difficult to pipette it off.

4. The Transgenic Core microinjects nuclease reagents into the pronucleus. This has been demonstrated to be superior to cytoplasmic injection for protein expression from mRNA (Wefers et al., 2013). The efficiency of pronuclear microinjection and cytoplasmic microinjection is the same (for example the data in Table S1 of Yang et al. shows that for every 100 embryos undergoing pronuclear microinjection with 5 ng/ul Cas9 mRNA + 2.5 ng/ul sgRNA + 10 ng/ul Oct4-GFP donor DNA that 13.3 genetically modified embryos were produced. For every 100 embryos undergoing cytoplasmic injection with 100 ng/ul Cas9 mRNA + 50 ng/ul sgRNA + 200 ng/ul Oct4-GFP donor DNA 13.6 genetically modified embryos were produced.)

5. Mix together nucleic acids in 0.02 um filtered RNAse Free Microinjection Buffer at the desired concentrations. Prepare fifteen aliquots of 50 ul in 1.5 ml microtubes. Store at -80°C.

 

Suggested Concentrations

Cas9 mRNA

5.0 ng/µl

sgRNA

2.5 ng/µl

Oligonucleotide or Plasmid Donor

10 ng/µl

5’ TALEN

2.5 ng/µl

3’ TALEN

2.5 ng/µl

Oligonucleotide or Plasmid Donor

10 ng/µl

5’ ZFN mRNA

2.5 ng/µl

3’ ZFN mRNA

2.5 ng/µl

Oligonucleotide or Plasmid Donor

10 ng/µl

 

Bibliography

Geurts AM, Cost GJ, Remy S, Cui X, Tesson L, Usal C, Menoret S, Jacob HJ, Anegon I, Buelow R. 2009. Generation of Gene-Specific Mutated Rats Using Zinc-Finger Nucleases. Methods in Molecular Biology. 597: 211- 225.

Montigny WJ, Phelps SF, Illenye S, Heintz NH. 2003. Parameters influencing high-efficiency transfection of bacterial artificial chromosomes into cultured mammalian cells. Biotechniques. 35:796-807.

Wefers B, Panda SK, Ortiz O, Brandl C, Hensler S, Hansen J, Wurst W, Kühn R. 2013. Generation of targeted mouse mutants by embryo microinjection of TALEN mRNA. Nat Protoc. 8:2355-2379.

Yang H, Wang H, Shivalila CS, Cheng AW, Shi L, Jaenisch R. 2013. One-Step Generation of Mice Carrying Reporter and Conditional Alleles by CRISPR/Cas-Mediated Genome Engineering. Cell. 154:1370-1379.